Methylotrophic yeasts are known to provide suitable expression systems for heterologous proteins. Hansenula polymorpha and Pichia pastoris, for example, are easy to handle host organisms for a wide variety of foreign genes (for reviews see Gelissen & Melber 1996 and Cregg & Madden 1988). The promoters for the enzymes involved in methanol metabolism in these organisms are particularly strong, and these promoters are generally used to control the heterologous expression of proteins (see EP 0173 378, EP 0299 108, EP 0183 071).
The specific carbon source used for cultivation of these organisms has an enormous influence on the regulation of methanol metabolism promoters. Until now, methanol and glycerol have been considered adequate substrates for the methylotrophic yeast expression system, while glucose has been considered inadequate (EP 299 108). During growth on methanol, key enzymes of methanol metabolism in Hansenula polymorpha are present in large amounts (Gelissen et al. (1994). Similarly, during growth on glycerol significant levels of the key enzymes methanol oxidase (MOX) and formate dehydrogenase (FMDH) can be detected. Derepression of the MOX and FMDH promoters permits expression of MOX and FMDH during growth on glycerol. However, these enzymes of methanol metabolism are absent in batch cultures if glucose is used as the carbon source (see Gelissen & Melber and EP 0299 108) (in glucose limited chemostat cultures of Hansenula polymorpha and Kloeckera sp. 2201, FMDH is only produced below growth rates of 0.1 h.sup.-1 in very small amounts (Egli et al. 1980)). Thus glucose is considered a repressor of the methanol metabolism promoters. These promoters permit only very limited expression of their genes when glucose is the available environmental carbon source.
Therefore, so far nonrepressive carbon sources, e.g. glycerol or methanol, are the standard carbon sources for the heterologous expression of proteins in methylotrophic yeasts. Hansenula polymorpha is cultivated for the production of a wide variety of pharmaceutical proteins in a fed batch process either in the single carbon source mode with glycerol or in the two carbon source mode with glycerol and additional methanol (Gelissen & Melber 1996). The common process strategy is described in more details by Weydemann et al. (1995) for the production of hirudin. A process for the production of thrombomodulin with a Pichia pastoris strain using also glycerol as a non repressive carbon source is described by Chen et al. (1996).
However, due to the high price of glycerol this process is not cost-effective for the production of low cost proteins like feed or industrial enzymes. Methanol is not a useful alternative due to handling problems caused by its toxicity and volatility. Therefore in the production of low cost proteins, methylotrophic yeast systems are not typically used. Glucose-metabolizing filamentous fungi are the standard expression system. However, these fungi require complex media, form a viscous culture difficult to handle, and produce significant quantities of undesired proteins such as proteases, decreasing yield and making purification of the target protein awkward. Thus the methylotrophic yeast system would be a much preferable production method for low cost proteins if expensive or dangerous substrates were not required.